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Congratulations on the acceptance of the manuscript.
[# PeerJ Staff Note - this decision was reviewed and approved by Konstantinos Kormas, a PeerJ Section Editor covering this Section #]
All formatting issues resolved. Nomenclature corrected. Professional English throughout.
Methods now reproducible
Data robust, conclusions well-supported. Statistical analyses appropriate. Novel insights into microbiome dynamics during disease progression.
Significant improvements since last revision. The study provides valuable data on L. chuanxiong root rot microbiome shifts and identifies promising biocontrol candidates. Recommend ACCEPTANCE.
Dear Dr. Zheng,
Please carefully address the comments of the single reviewer. Please perform complete answers to all points highlighted by this reviewer,
Best regards
Rodrigo
While the manuscript is written in proficient English, there are significant and persistent issues with clarity, precision, and professional reporting standards that were not adequately addressed in this revision.
A pervasive lack of care is evident throughout the manuscript, starting with the title, where the fungal species names (Fusarium oxysporum and F. solani) are still not italicized. This fundamental formatting error must be corrected consistently across the entire manuscript (headings, text, tables, figures).
The revised text is now filled with numerous distracting formatting errors, such as "Figure 4Fig. 2," "Figure 1aFig. 3a-c," and "Figure 6a8a-c." These errors suggest a careless revision process and significantly hinder readability. A thorough proofread is required to present the work in a professional manner.
The experimental design suffers from fundamental flaws because critical components remain unvalidated and supporting evidence is presented in an unacceptable manner. The revisions have failed to rectify these core issues.
The authors' attempt to fulfill Koch's postulates is undermined by the unacceptable quality of their evidence. The newly provided Supplementary Figure 1 is scientifically unusable. It presents 12 unlabeled micrograph images with no indication in the figure or its legend as to which images correspond to F. oxysporum and which to F. solani. As it stands, this figure provides zero evidential value and fails to support the claim of morphological verification. This is a severe lapse in basic scientific reporting.
The authors have again failed to provide experimental validation for their surface sterilization protocol. Citing established methods is not a substitute for performing a standard and essential control experiment, such as plating the final rinse water or making tissue imprints onto an agar medium. Without this validation, the central claim that the identified microbial communities are truly "endophytic" remains unsubstantiated and speculative.
Due to the significant and unresolved flaws in the experimental design and reporting, the validity of the study's main findings and conclusions is severely compromised. The link between the data and the interpretations is, in many places, tenuous.
The critical point raised in the previous review regarding the outdated nomenclature of Fusarium solani (which should be updated to Neocosmospora solani) has been completely ignored. This is not mentioned in the response letter, and no changes have been made in the manuscript. The use of current and accurate taxonomy is non-negotiable for a scientific publication. Ignoring a direct and valid scientific correction demonstrates a serious lack of scholarly diligence.
Since the disease etiology has not been rigorously established with clear evidence, and the endophytic nature of the microbes is unconfirmed, any conclusions regarding "protective responses" or "pathogenic tendencies" are entirely correlational. The data show a shift in microbial populations in unhealthy-looking tissue, but cannot, in its current form, prove causation or a specific host-pathogen dialogue.
This study has the potential to be interesting, but it is currently framed around a narrative that is not supported by the necessary experimental rigor. The authors' revisions were insufficient, and their disregard for certain key corrections is concerning.
For this manuscript to become scientifically sound and publishable, the following fundamental issues must be addressed:
The most critical step is to provide clear, interpretable, and properly labeled evidence that validates the fulfillment of Koch's postulates. This requires remaking Supplementary Figure 1 with unambiguous labels and a descriptive legend.
The authors must either provide clear experimental evidence of effective surface sterilization (via controls) or reframe their entire discussion to be about "tissue-associated" microbes, removing the strong and unsupported claims about "endophytes."
The authors must update the fungal nomenclature to reflect current taxonomic standards (Neocosmospora solani) and conduct a meticulous proofread of the entire manuscript to correct all formatting errors, starting with the title.
These points are non-negotiable. The manuscript requires a thorough and thoughtful revision, not just a superficial one.
Dear Authors,
Thank you for submitting your manuscript to PeerJ. After thorough evaluation of the submission and consideration of the detailed comments from two expert reviewers, the editorial decision is Major Revision. The study presents a valuable dataset characterizing microbiome changes during root rot progression in Ligusticum chuanxiong, with the potential to contribute to the understanding of plant-microbiome interactions. However, both reviewers identified significant conceptual and methodological issues that must be addressed before the manuscript can be considered for publication.
Below, we summarize the key points requiring your attention:
1. Establishment of Disease Etiology (Critical)
Reviewer Concern: The central claim of disease progression is unsubstantiated without a clear identification of the causal pathogen(s). The manuscript frequently refers to "rot," but does not rigorously define the pathology or demonstrate causality.
Please provide etiological confirmation to substantiate the claim that the observed microbial shifts are associated with a defined disease process. This may include the isolation and morphological characterization of potential pathogens, molecular identification using PCR or qPCR targeting known agents such as Fusarium spp. or Pectobacterium, and—if feasible—pathogenicity assays that fulfill Koch’s postulates. If conducting these experiments is not currently feasible, we recommend that the manuscript be reframed as a descriptive observational study. In this case, the narrative should focus on microbiome variation associated with symptomatic versus asymptomatic rhizomes, while avoiding any strong causal claims regarding disease progression, pathogenicity, or host response mechanisms.
2. Validation of Endophyte Status
Reviewer Concern: The protocol used for surface sterilization is not validated, casting doubt on the claim that microbes are truly endophytic.
Please provide experimental validation of the surface sterilization protocol to support the claim that the microorganisms identified are truly endophytic. This could include procedures such as plating the final rinse water, performing tissue imprint tests, or other standard sterility controls.
If such validation is not available or was not performed, we recommend that all references to “endophytes” or “endophytic communities” be carefully revised throughout the manuscript. Instead, please refer to them more cautiously as “microbial communities associated with surface-sterilized tissue”, in order to accurately reflect the limitations of the current methodology.
3. Clarification of Tissue Type and Disease Symptomatology
Reviewer Concern: The manuscript refers to “root rot,” while samples were taken from rhizomes. Also, terms like “rot layer” are used without pathological classification (e.g., soft/wet/dry rot).
Please ensure that precise anatomical terminology is used throughout the manuscript. For example, refer to the sampled organ as a rhizome rather than a “root” where appropriate. In addition, the disease symptoms should be described more clearly and in greater detail, including visual indicators (e.g., lesions, discoloration), texture changes (e.g., sponginess or softening), color differences, and the presence of any distinctive odors associated with decay. Finally, please review and revise all relevant sections of the manuscript and figure legends to ensure consistent use of these corrected terms and descriptions across the entire text.
4. Functional Inference Limitations
Reviewer Concern: The use of FUNGuild and PICRUSt2, while informative, does not justify strong functional conclusions given the taxonomic resolution (genus-level) and lack of strain-level validation.
Please temper the language used when discussing functional predictions derived from tools such as PICRUSt2 and FUNGuild, particularly where strong conclusions are drawn about microbial roles in pathogenicity or plant protection.
Additionally, the Discussion section should explicitly acknowledge the limitations of these functional inferences, especially given that marker-gene-based predictions lack strain-level resolution and may not accurately reflect the actual metabolic capabilities or ecological functions of the detected taxa.
5. Co-occurrence Network Interpretation
Reviewer Concern: Co-occurrence networks are complex but offer limited additional insight. The biological relevance of increased positive correlations or edge density is not sufficiently discussed.
Please clarify the biological insights provided by the co-occurrence networks beyond what is already demonstrated by the alpha and beta diversity analyses. Clearly explain the additional value these networks bring to the interpretation of microbiome shifts during disease progression. in addition, the Discussion should consider alternative interpretations of the observed network patterns. For example, the increase in positive correlations could reflect opportunistic proliferation of multiple taxa in decaying tissue rather than functional synergy or coordinated ecological interactions.
6. Abstract and Title Revisions
Reviewer Suggestion (R2):
Clearly mention known pathogens (Fusarium oxysporum, F. solani, etc.) if etiological work supports it.
State the novelty and potential applications of the findings (e.g., support for biocontrol).
Ensure terminology is consistent: rhizome vs. root; healthy layer vs. transitional tissue.
7. Additional Revisions
Include amplification product size and target region for both primer sets used.
Use correct scientific formatting (e.g., italicizing species names).
Improve figure legends, e.g., Figure 5c is labeled “phylum” but shows genera.
Provide images of disease symptoms and clearly labeled sample types if available.
If proposing microbial candidates for synthetic communities, provide justification based on specific ecological or functional traits.
Conclusion and Next Steps
The manuscript addresses an important topic and contains rich microbial data. However, the interpretive framework currently outpaces the experimental support. A revised manuscript that either:
provides the missing pathological and methodological validation, or
presents the findings as descriptive and exploratory,
will significantly strengthen its scientific rigor.
We encourage the authors to revise the manuscript accordingly and provide a detailed point-by-point response to each reviewer comment and editorial request.
While the manuscript is written in proficient English, there are significant issues with clarity and precision in its core concepts and reporting.
The study uses "root rot" in the title and throughout, but the sampling was performed on rhizomes. These are distinct organs, and this lack of precision should be corrected for accuracy.
The term "rot" is used as a key experimental variable, yet it is not defined from a plant pathology perspective. Is this wet rot, dry rot, or soft rot? What are the specific visual and physical characteristics that define the "diseased rhizome-rot layer (DRR)"? This needs to be explicitly described.
There is a critical error in the legend for Figure 5c. It is described as "differential phyla of endophytic fungi," but the figure clearly shows data at the genus level. This needs to be corrected.
I have fundamental concerns regarding the experimental design, which currently prevent the substantiation of the study's main claims. The design lacks the necessary pathological grounding to establish a causal link between the observed microbial communities and a specific disease.
The entire study is built on the premise of a "disease progression," but the etiological agent is never identified. There is no information on whether the symptoms observed in the field were caused by a fungus, a bacterium, a complex of organisms, or even abiotic factors. Without identifying the primary pathogen(s) through standard pathological methods (e.g., isolation, microscopy, molecular identification, and ideally, fulfilment of Koch's postulates), the study is limited to a descriptive comparison of microbial communities from healthy vs. unhealthy-looking plants. This fundamentally undermines the entire narrative of a host-pathogen interaction and renders the term "disease progression" speculative.
The claims about "endophytic" communities are central to the paper's conclusions. However, the methods section fails to describe any control experiments to verify the efficacy of the surface sterilization protocol. How can the authors be certain that the DNA extracted was not contaminated by epiphytic microbes? Standard controls, such as plating the final rinse water or tissue imprints, are necessary to validate claims about endophytes. Without this validation, the communities should be more cautiously referred to as "microbial communities from surface-sterilized rhizome tissues." The current approach does not meet the standard for rigorous investigation in this area.
Due to the significant flaws in the experimental design mentioned above, the validity of the study's main findings and conclusions is severely compromised. The link between the data and the interpretations is, in many places, tenuous.
Since the cause of the disease is unknown, any conclusion regarding "protective responses" or "pathogenic tendencies" of specific microbial taxa is entirely correlational and speculative. The observed shifts could be a consequence of general plant tissue decay rather than a specific response to a pathogen. The data shows a correlation, not causation.
The functional predictions from PICRUSt2 and FUNGuild (Figure 3) are presented with little caution. It is well-known that function, especially pathogenicity, can be highly strain-specific. A genus like Fusarium contains both virulent pathogens and harmless saprophytes. Therefore, inferring community function from marker-gene data in a system with an unknown etiology is not reliable.
The co-occurrence networks (Figures 6 and 7) are complex but not particularly informative. They do not appear to reveal insights beyond what is already evident from the simpler differential abundance analyses. For example, the increase in positive correlations could simply be an artifact of several opportunistic microbes proliferating together in dying tissue. These figures do not currently add sufficient value to justify their inclusion.
In short, the conclusions are not adequately supported by the evidence because the foundation of the experiment—the "disease"—is undefined.
This study addresses a potentially interesting ecological question, and a significant amount of sequencing data has been generated. However, as outlined above, the work is currently framed around a narrative of disease progression that is not supported by the experimental design. The lack of basic plant pathology work is a critical omission that prevents the data from being interpreted in the context of host-pathogen interactions.
For this manuscript to become a scientifically sound contribution, the following fundamental issues must be addressed:
Establish the etiology, the most critical step is to identify the primary causal agent(s) of the root/rhizome rot in your study system. This is non-negotiable for a study claiming to investigate disease progression.
You must either provide clear evidence of effective surface sterilization (via controls) or reframe your discussion to be about "tissue-associated" microbes, removing the strong and currently unsupported claims about "endophytes."
I see two potential paths for this manuscript:
1. Conduct the necessary pathological work to identify the etiological agent(s). This would validate your experimental system and allow you to interpret your existing data within a robust framework.
2. If new experiments are not feasible, you must completely reframe the manuscript. It would have to be presented as a purely descriptive, preliminary study of microbial communities in healthy vs. decaying rhizomes of L. chuanxiong from a specific field. All strong claims regarding "disease," "pathogenesis," "protection," and "host response" must be removed. The manuscript would have a much more limited scope, but it would be a more honest representation of the data.
The author has selected a novel topic and presented valuable findings, with the literature included systematically and information arranged chronologically, which will help pave the way for future research in this area.
The experiment was conducted in a systematic and statistically sound manner, though some parts are somewhat difficult to understand
The data are presented with clarity, using meaningful charts and figures.
The author should be appreciated for presenting significant findings related to the microbial community associated with different stages of root rot disease in Ligusticum chuanxiong. However, several areas require improvement to enhance the clarity and strength of the manuscript:
It lacks specific information about the causal organism(s) responsible for root rot in the abstract. The abstract should clearly mention the pathogen(s) involved. Additionally, the novelty and significance of the findings should be explicitly stated at the end of the abstract.
While the manuscript suggests the potential use of beneficial microbes for constructing a synthetic microbial community, it is essential first to analyze the biochemical characteristics of the culturable microorganisms. Furthermore, it should be considered whether microbial metabolites influence the activity or metabolism of other microbes in the community.
Detailed descriptions of disease symptoms at different stages of root rot are crucial. Including this information would greatly strengthen the manuscript by providing a clearer understanding of disease progression.
1. Kindly mention which pathogen(s) incite root rot in Ligusticum chuanxiong.
2. Please consider including numerical data to support the findings wherever applicable.
3. At the end of the abstract, include a statement on the novelty of the work, for example: "The study identifies potential microbial candidates that play a dominant role in maintaining plant health."
4. It is recommended to include the common name of the medicinal plant (L. chuanxiong) for broader reader clarity.
5. Kindly list the specific pathogens responsible for root rot in L. chuanxiong, along with references if available.
6. Please clarify whether there is a specific rationale for selecting the particular plant part (e.g., rhizome, root zone) used in this study.
7. If applicable, discuss the practical application of this research, i.e., whether the beneficial microbes identified are compatible and can be developed for field use.
8. Indicate whether the beneficial microbes studied are mutually compatible and can coexist functionally in a synthetic microbial community.
9. Please specify the major root rot pathogens identified in the diseased samples.
10. During sample collection, indicate the age or growth stage of the plants—especially differentiating between early infection and advanced (decayed) stages.
11. The manuscript would be strengthened by including disease symptoms corresponding to different stages of infection or by presenting images of longitudinally dissected rhizomes.
12. Kindly mention the amplification product size (bp) and the target region for both primer sets used (e.g., 16S rRNA, ITS).
13. In the figures, please highlight the dominant OTUs using distinct colors or legends to improve clarity.
14. Revise sentence constructions where necessary; some lines begin abruptly and lack context.
15. Ensure that scientific names are formatted correctly, with genus names starting with a capital letter and species names in lowercase (e.g., Bacillus subtilis).
16. Include a description of signature metabolites present in the root exudates that are involved in recruiting beneficial microbial communities.
17. Consider discussing whether microbial metabolites produced by the beneficial microbes contribute to biochemical pathways that facilitate site-specific symbiotic microbial core community formation for plant growth promotion and disease resistance.
18. Specifically describe the OTU diversity and relative abundance of important biocontrol agents such as Bacillus spp. and Trichoderma spp.
19. If the study aims to construct a synthetic microbial community, please list the microbial candidates selected and provide justification for their inclusion.
20. Finally, consider discussing any potential fungal bioagents identified in the study, along with their role in plant health or disease suppression.
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